DOI: 10.1111/exd.12535
Original Article
www.wileyonlinelibrary.com/journal/EXD
Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells Michal Sarna1,2, Andrzej Zadlo2, Pawel Hermanowicz3, Zbigniew Madeja4, Kvetoslava Burda1 and Tadeusz Sarna2 1 Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Krakow, Poland; 2Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 3 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 4Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Correspondence: Michal Sarna, Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Mickiewicza 30, Krakow 30-059, Poland, Tel.: (+48) 12 6172984, Fax: (+48) 12 6340010, e-mail: [email protected]
Abstract: The relationship between melanin pigmentation and metastatic phenotype of melanoma cells is an intricate issue, which needs to be unambiguously determined to fully understand the process of metastasis of malignant melanoma. Despite significant research efforts undertaken to solve this problem, the outcomes are far from being satisfying. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells. This prompted us to examine the mechanical effects of melanosomes on the transmigration abilities of melanoma cells. Here, we show for the
first time that melanin granules inhibit transmigration abilities of melanoma cells in a number of granules dependent manner. Moreover, we demonstrate that the inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may play a key role in the efficiency of melanoma cells spread in vivo. Our findings may also contribute to better understanding of the process of metastasis of malignant melanoma.
Introduction
knowledge would contribute to better understanding of metastasis of melanoma and could even lead to the development of a more advanced diagnosis of this malignant tumor. Metastasis is a complex process in which cancer cells spread from the primary tumor to colonize distant organs (11). One of the most critical steps in the process of metastasis is the invasion of host tissues (12). During this stage of metastasis cancer cells encounter multiple barriers inside the tissues (13,14), with the most significant being the endothelial barrier (15). To pass through different barriers cancer cells must be able to undergo extensive deformation of their cellular body (16). This unique mechanical property of cancer cells depicted as elasticity is a result of significant changes in the cytoskeleton of the cells and is acquired during cancer transformation (17,18). Consequently, cancer cells become very soft and highly deformable, which is believed to be one of the main reasons for their ability to transmigrate through multiple barriers highly efficiently (19,20). Different studies have demonstrated that cancer cells with lower values of the Young’s modulus (the measure of elasticity) also exhibit an increased invasive potential (21–24). Therefore, cell elasticity is viewed as an important indicator of the metastatic phenotype of cancer cells. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells (25,26). This has led us to formulate the hypothesis that melanosomes could significantly affect the transmigration abilities of melanoma cells and, as a result, they could affect the cells invasiveness. Because of obvious importance, the hypothesis
Among cellular properties of different cancer types, the ability of melanoma to synthesize melanin clearly stands out. Melanin in pigmented cells, including melanoma cells is present in the form of distinct granules called melanosomes (1). These organelles display a range of different shapes and sizes (2) and have unusual mechanical properties (3). Melanoma originates from transformed melanocytes – cells that normally produce melanin in the skin (4). However, unlike melanocytes, which continuously transfer the synthesized pigment to keratinocytes, effectively decreasing the number of melanosomes inside the cells; melanoma cells do not excrete the pigment, which may lead to heavy pigmentation of the cells (5). This unique property of melanoma cells puzzled researchers for decades, and still remains unclear. Under normal conditions melanin synthesis is regulated by different factors and has protective function (6). On the other hand, when melanogenesis is deregulated (such as in melanoma), it can have a detrimental effect (7–9). In spite of significant research efforts, the relationship between melanin pigmentation and metastatic phenotype of melanoma cells remains controversial. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. It is important to realize that melanoma is one of the deadliest types of cancer with poor prognosis when diagnosed in the metastatic stage (10). Therefore, it is of key importance to determine the relationship between melanin pigmentation and metastatic phenotype of melanoma cells. Such
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
Key words: cell elasticity – invasion – melanin pigmentation – melanoma – metastatic phenotype
Accepted for publication 3 August 2014
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should be verified experimentally. It is important to realize that mechanical effects of melanin granules on melanoma cells invasive abilities have not been studied by other researchers and remain unknown. In this work, we show for the first time that melanin granules inhibit the transmigration abilities of melanoma cells through artificial barriers in vitro. Moreover, we demonstrate that the inhibitory effect of melanosomes depends on the number of granules in the cells. On the other hand, our data indicate that the pigment granules have no significant impact on vital functions of pigmented cells including their tumorigenic properties. This has led us to conclude that the observed inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may be an important indicator of the metastatic phenotype of melanoma cells in vivo. Moreover, our findings may contribute to better understanding of the process of metastasis of malignant melanoma.
Methods Cell culture Human melanoma SKMEL-188 cells were cultivated in Ham’s F10 culture medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and maintained at 37°C in a 5% CO2 humidified atmosphere. Under these conditions SKMEL-188 cells did not synthesize melanin and such cells were used in the analysis as control non-pigmented melanoma cells. Different levels of melanin pigmentation of the cells were obtained using a non-invasive method for melanin synthesis as described elsewhere (27). After this protocol, cells were passaged and maintained in Ham’s F10 culture medium for 48 h to ensure that cells did not synthesize melanin during the analysis and to minimize the risk of any shock to the cells caused by the exchange of culture media. All experiments described in this work were carried out in Ham’s F10 medium.
Melanin determination in cell samples Analysis of melanin content in the examined samples was determined by electron paramagnetic resonance (EPR) spectroscopy based on intensity and spectral parameters of characteristic EPR signals of eumelanin and pheomelanin (28). For EPR analysis, cells were detached from the culture dish, pelleted, counted, incubated in high concentration of zinc acetate, frozen and stored at 77 K. The number of cells for each sample was approximately 106 cells and final concentration of zinc ions was 50 mM. EPR examination was carried out in liquid nitrogen, using standard finger-type quartz dewar and EMX-AA spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at X-band with 100 kHz magnetic modulation. Detailed description of EPR analysis used in this work can be found elsewhere (29,30). The average number of melanin granules inside cells was determined by comparing double integrals of the EPR signals of cell samples with that of 2.05 mg/ ml synthetic cystein-L-dopa-melanin used as a standard and with the EPR signal of a known number of purified melanosomes isolated from pigmented SKMEL-188 cells run under identical experimental conditions. Based on relative intensities of these signals, an equivalent EPR signal of a single melanin granule was determined. Such standard was then used to calculate the average number of melanin granules per cell in cell samples. The number of melanin granules used as a standard in EPR analysis was approximately 2 9 108 granules.
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Elasticity analysis Mechanical analysis of the cells was conducted using Agilent 5500 atomic force microscope (AFM) (Agilent Technologies, Austin, TX, USA) coupled with top-down optics (Navitar, Rochester, NY, USA). During the analysis, cells were maintained in culture medium at 37°C. AFM measurements were performed using standard silicon nitride cantilevers (Bruker Probes, Karlsruhe, Germany) with nominal tip radius of 20 nm and with experimentally determined spring constants (31). Mechanical measurements were conducted in force spectroscopy mode that consists of measuring force-displacement curves (32). Force curves were collected at a rate of 1 Hz per curve from a ‘grid’ of 8 9 8 force curves applied onto the centre area of a cell. Before each grid was selected, a topography image of a cell was obtained in tapping ac mode, to precisely position the grid onto the centre of the cell body. During mechanical measurements, both the applied force and indentation depth were carefully monitored to prevent damage to the cells and to avoid possible substrate induced effects. Forty cells for each sample were analysed. Furthermore, to show a co-localization effect of melanin granules on the elastic properties of the cells, representative force maps of pigmented and non-pigmented cells were made. This was achieved from a grid of 128 9 128 force curves applied onto the entire scan area. Analysis of force curves was made using AtomicJ – our Open Source software (33). Briefly, the values of the Young’s modulus were estimated from force curves by converting force-displacement curves into force-indentation curves and fitting with the Sneddon model (34). During the analysis, the correction for thin samples was used to minimize any substrate generated artifacts (35). The half opening angle of the AFM tip was 25°, and the Poisson ratio of the cells was taken to be 0.5, which is typical for soft biological materials (36).
Transwell invasion assay Transmigration efficiency of the cells was determined using a well established Transwell assay for the analysis of the invasive abilities of cancer cells as described elsewhere (37). To highlight the mechanical aspect of the experiment, the membrane was neither coated with collagen and endothelial cells nor with Matrigel. For the analysis, cells were seeded into 6-well plates (three wells per condition) containing Transwell inserts (pore size: 8 lm; Corning Incorporated, Corning, NY, USA). After 24 h, cells were trypsinized and counted. The number of cells transmigrated through porous membrane was compared with the number of cells that remained on the top of the membrane. The assay was repeated three times for statistical analysis.
Proliferation analysis For proliferation analysis, cells were seeded into 12-well plates (one plate per condition) and maintained in culture for 24, 48, 72 and 96 h. After each time interval, cells were trypsinized and counted. The number of cells determined after 24 h for each experimental condition was the reference number of cells indicated as 100%. The assay was repeated three times for statistical analysis.
Migration analysis Migration activity of the cells was determined based on time-lapse monitoring of the movement of individual cells as described elsewhere (38). This was conducted using a fully automated Leica DMI6000 B (Leica Microsystems GmbH, Wetzlar, Germany)
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
Melanin granules inhibit melanoma invasion
inverted microscope with a dedicated incubation chamber. During the analysis, cells were maintained in culture medium at 37°C in a 5% CO2 humidified atmosphere. The movement of the cells was recorded for 8 h, with 5 min time intervals. Tracks of individual cells were determined from a series of changes in the cell centroid positions as described elsewhere (39). Trajectories of 40 cells for each condition were analysed for statistics.
Gelatin zymography assay Expression of matrix metalloproteinase was determined using standard protocol for gelatin zymography as described elsewhere (40). Prior to the analysis, cells were incubated in serum-free culture medium for 24 h, after which the supernatants were collected and equivalent amounts of protein for each sample were diluted in sample buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol and 0.4% bromophenol blue). Samples were electrophoresed on SDS-PAGE gels containing 1% gelatin. After electrophoresis, gels were washed in 2.5% Triton X-100 for 30 min to remove SDS. Subsequently, gels were placed in substrate buffer (50 mM Tris pH 7.7, 5 mM CaCl2 and 0.02% NaN3) and incubated for 40 h at 37°C under gentle agitation. After incubation, gels were stained in 40% methanol, 7% glacial acetic acid solution containing 0.4% Coomassie Brilliant Blue R250 for 45 min and destined in the same solution without Coomassie Brilliant Blue for 30 min. The assay was repeated on cell samples from three independent cultures. Densitometric analysis of the gel bands was made using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Results Figure 1 shows EPR spectra of human melanoma cells containing different amounts of melanin pigment and non-pigmented melanoma cells. These results clearly demonstrate that only pigmented cells (Fig. 1a–c) exhibit strong EPR signal with magnetic parameters typical for melanin free radicals (41). The pigment in these cells is a mixture of pheomelanin and eumelanin, which is common for human melanoma cells (42). On the other hand, no EPR signal of melanin radicals is detected in non-pigmented melanoma cells (Fig. 1d). Figure 1 also shows EPR spectra of synthetic cysteine-L-dopa-melanin and purified melanin granules isolated from melanoma cells used as standards in EPR analysis (Fig. 1e and f). The average number of melanin granules inside cells determined by EPR spectroscopy is shown in Table 1. Figure 2 shows results obtained with AFM and demonstrate the impact of melanin granules on the elastic properties of melanoma cells. These results show histograms of Young’s modulus values for cells containing different number of melanin granules (Fig. 2a–c) and non-pigmented melanoma cells (Fig. 2d). As evident from the data, cells containing more melanosomes exhibit higher values of the Young’s modulus. Moreover, the log-normal distribution of Young’s modulus values for pigmented cells becomes more pronounced as the number of melanosomes inside cells increases. Similar effect was observed in our previous studies
Confocal microscopy Immunofluorescence analysis was performed on cells fixed with 3.7% formaldehyde, permeabilised with 0.1% Triton X-100, blocked with 3% BSA and stained with mouse monoclonal antibody against human microtubule, followed by Alexa Fluor 488 goat anti-mouse IgG, with TRITC-phalloidin and Hoechst 33342 (Sigma-Aldrich), respectively. Images were obtained using Nikon Ti-E A1 inverted microscope coupled with laser scanning module equipped with: 405, 488, 561 and 638 nm laser diodes, 4 PMT detectors for fluorescence and 1 PMT detector for transmission (Nikon Instruments, Tokyo, Japan). Images were analysed using NIS-Elements AR 3.1 software (Nikon).
Statistical analysis Statistical significance of differences in mean values was assessed using two-sample independent Student’s t-test at the 95% confidence level. Differences among means were reported using approximated P values. Statistical analysis was made using Mathematica 8.0 software.
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Figure 1. Determination of melanin content inside cells. Electron paramagnetic resonance (EPR) spectra of melanoma cells with different amounts of melanin pigment (a–c) and non-pigmented control cells (d). The EPR signal of melanin in pigmented cells is a superposition of a pheomelanin signal (lower field component) and an eumelanin signal (higher field component). EPR spectra of synthetic cysteine-L-dopa-melanin (e) and purified melanin granules isolated from melanoma cells (f) that were used as standards in the determination of the average number of melanin granules per cell in cell samples. Signal intensities (y-axis) were normalized to maximum amplitude to better show the contribution of pheomelanin component in the spectra.
Table 1. Transmigration efficiency coupled with elastic properties of human melanoma cells versus number of melanin granules inside the cells Degree of cell pigmentation
Number of melanin granules1
Young’s modulus (kPa)2
Non-pigmented Lightly pigmented Moderately pigmented Heavily pigmented
– 22 2 42 3 73 4
1.97 3.23 4.91 6.97
0.05 0.85* 1.22** 1.56***
Transmigration efficiency (%)3 18.79 13.83 9.18 3.94
0.51 0.76* 0.46** 0.27***
Statistical significance between mean values for both Young’s modulus and transmigration efficiency is depicted as the following: *statistically significant versus control cells, **statistically significant versus lightly pigmented cells and ***statistically significant versus moderately pigmented cells. For all values P < 0.0001. Average number of melanin granules per cell (mean SD) determined by EPR spectroscopy. 2 Average values of Young’s modulus (mean SD) determined based on function fit to the data in the histograms. 3 Transmigration efficiency represents the average number of cells (mean SD) transmigrated through porous membrane determined based on a Transwell assay (n = 9 for each sample). 1
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
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Figure 2. Impact of melanin granules on the elastic properties of melanoma cells. Histograms of Young’s modulus values for cells with different number of melanin granules: (a) 22 2 melanosomes, (b) 42 3 melanosomes, (c) 73 4 melanosomes and non-pigmented control cells (d) no granules were determined in this sample. Data for pigmented cells were fitted with log-normal functions whereas data for non-pigmented cells was fitted with Gaussian function. Forty cells for each condition were analysed with 64 measurement points on a single cell (n = 2560 for every sample). Representative atomic force microscope (AFM) amplitude images of pigmented (e) and non-pigmented (f) melanoma cells and corresponding force maps of Young’s modulus values (g, h). Insets in the images show optical preview of the cells during AFM analysis. Scale bars in the images represent 10 lm. Colour bars in force maps represent Young’s modulus values, range: 0–50 kPa.
(25,26) and discussed in detail. Numerical values of Young’s modulus together with the number of melanin granules inside cells are shown in Table 1. It should be emphasized that during AFM analysis, elasticity measurements were taken from the central region of the cell to avoid possible artifacts when going ‘away’ from the centre of the cell towards cell edges (43). The cell central body is also the most critical area of cancer cells when considering transmigration trough artificial barriers in vitro or different barriers in vivo. This is because the cell central body (the thickest part of a cell) must undergo significantly higher deformation, when compared to outer regions of the cell such as thin lamellipodia (44–46). Figure 2 also shows representative AFM images (Fig. 2e and f) and corresponding force maps (Fig. 2g and h) of pigmented and non-pigmented melanoma cells, respectively. Insets in the images clearly show that melanin granules surround the nucleus of a pigmented cell. As evident from force maps, the values of the Young’s modulus are significantly higher around the cell nucleus (at the locations of the melanosomes) when compared to non-pigmented control cell. It should be emphasized that melanin granules did not modify the topography of pigmented cells when compared to non-pigmented cells. Figure S1 shows height images and corresponding profiles of melanoma cells with different number of melanosomes and non-pigmented melanoma cells. As evident from the data, pigmented and non-pigmented cells exhibit similar topography. Transmigration efficiency of the cells determined from a Transwell assay is shown in Table 1. These results demonstrate that cells containing more melanin granules are less likely to transmigrate through membrane pores compared to cells without melanosomes. The data clearly shows that melanin granules exhibit an inhibitory effect on the transmigration abilities of human melanoma cells and that the effect depends on the number of granules inside the cells. To determine whether the observed inhibitory effect of melanosomes on the transmigration abilities of melanoma cells was solely mechanical in nature, we examined the impact of melanin
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Figure 3. Impact of melanin granules on melanoma cell vital functions and the cell cytoskeleton. (a) Column graph showing proliferation abilities of pigmented and non-pigmented melanoma cells. Results obtained from three independent experiments (n = 9 for each sample). (b) Column graph showing migration activity of pigmented and non-pigmented melanoma cells based on the movement of individual cells (n = 40 for each sample). (c) Representative gel is from three zymographies carried out on independent cell cultures (n = 3 for every sample) and shows the expression of pro-MMP-9 for pigmented and non-pigmented melanoma cells. Numbers on the gel refer to the level of melanin pigmentation of the cells: 0 – non-pigmented control cells, 1 – lightly pigmented, 2 – moderately pigmented and 3 – heavily pigmented. Representative confocal microscopy images of actin (red), microtubules (green) and nucleolus (blue) for pigmented (d) and nonpigmented (e) melanoma cells. Overlay images of scattered laser light and fluorescence for pigmented (f) and non-pigmented (g) melanoma cells show melanin granules in pigmented cell (indicated with arrows) surrounding the cell nucleus. No granules are present in non-pigmented cell. Scale bars in the images represent 10 lm.
granules on the cell vital functions. Figure 3 shows the impact of melanosomes on proliferation abilities (Fig. 3a), followed by migration activity (Fig. 3b) and expression of metalloproteinase (Fig. 3c) in pigmented and non-pigmented melanoma cells. Based on quantitative analysis of the cell proliferation abilities and migration activity, we can conclude that melanin granules inhibit neither proliferation nor motility of pigmented melanoma cells in any significant way when compared to non-pigmented melanoma cells. Moreover, densitometric analysis of gels obtained from zymography assay revealed no significant differences in the expression levels of pro-matrix metalloproteinase-9 (pro-MMP-9) between cells containing different number of melanin granules and non-pigmented control cells (data not shown). Although human melanoma cells can express different matrix metalloproteinases (47), the MMP-9 is viewed as one of the most important metalloproteinases engaged in the formation of metastatic tumors (48). Figure 3 also shows the impact of melanin granules on the cell cytoskeleton. Representative fluorescence images of actin and microtubules for pigmented and non-pigmented melanoma cells are shown in Fig. 3d and e. These images reveal that melanin granules do not modify the cell cytoskeleton in any significant way. Overlay images of scattered laser light and fluorescence for pigmented and non-pigmented melanoma cells, respectively (Fig. 3f and g), clearly show that melanin granules in pigmented cell are mostly localized around the cell nucleus. We believe that such distribution of melanin granules inside melanoma cells is one the main reasons of the reduced capability of pigmented melanoma cells to pass through narrow barriers. The hard, nondeformable melanosomes (3) that are present in the most critical
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
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area of melanoma cells prevent the cells to undergo extensive deformation of their cellular body necessary for passage through small membrane pores. As evident from elasticity measurements taken from the cell central region, cells containing melanin granules show higher values of the Young’s modulus, which translates to reduced deformation capabilities of the cells when compared to non-pigmented control cells. Thus, in pigmented melanoma cells, melanin granules act as a natural inhibitor of the cells transmigration abilities.
Discussion In this work, we have demonstrated for the first time that melanin granules inhibit the abilities of melanoma cells to transmigrate through narrow barriers in vitro, in a number of granules dependent manner. Moreover, we have determined that the inhibitory effect of melanosomes is mechanical in nature being due to a dramatic change in the elastic properties of the cells caused by the presence and distribution of the pigment granules inside the cells. It is important to stress that the presence of melanosomes in melanoma cells did not affect any of the cell vital functions, including their proliferation activity, their motile abilities and expression of the key metalloproteinase (MMP-9). All these factors play a crucial role in the process of metastasis (49). Therefore, the results obtained in this study support the validity of our experimental model for melanoma cells invasive abilities, that is the clean, uncovered version of a Transwell assay. It should be stressed that the main purpose of our work was to demonstrate the mechanical aspect of the observed phenomenon in a quantitative manner. This goal would be very difficult to achieve, if possible at all, using a membrane coated with both collagen and endothelial cells or coated with Matrigel. The latter models are more suitable to examine other properties of cancer cells important for invasion as demonstrated elsewhere (50,51). Our experiments employing melanoma cells with different numbers of melanin granules allowed us to analyse, in non-invasive manner, cells with different elastic properties and, to correlate the elasticity of the cells with their transmigration efficiency. The obtained results confirm the widely accepted paradigm that elasticity is a unique indicator of the invasive abilities of different cancer cells, including melanoma cells. There is no doubt that elasticity is just one of many properties of cancer cells that play an important role during metastasis (52). However, taking into consideration the magnitude of the effect observed in this study together with the fact that melanoma cells can contain large numbers of melanosomes (5); we can conclude that at least under some circumstances, elasticity of melanoma cells could be the dominant factor responsible for the efficient spreading of the cells inside host tissues. Recently, using intra-vital imaging Pinner et al. demonstrated that pigmented melanoma cells displayed in vivo a lower metastatic phenotype then non-pigmented melanoma cells. Moreover, the authors reported that the cells could switch between high and low metastatic phenotype. Such switch was accompanied by changes in the cells pigment state (53). The authors attributed the observed results to the level of cell differentiation. Hence, pigmented melanoma cells are considered to be more differentiated and therefore less invasive (54). However, in their experimental model the authors used different growth factors and stimuli for the induction or inhibition of melanin pigmentation in cells. It cannot be ruled out that the used factors had a signif-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
icant impact on the level of cells differentiation. It should be emphasized that in our study, we did not use any growth factors or stimuli that would enhance the level of cells differentiation in any significant way. This allowed us to obtain melanoma cells with different amount of melanin pigment and to correlate the level of cell pigmentation with their transmigration abilities. It is also important to realize that melanin is not susceptible to enzymatic degradation (55), and the only way to reduce its content in melanoma cells is to stop synthesis of melanin. With continuous divisions, such cells will become less pigmented. However, this depigmentation process takes time and is not instantaneous. It is a matter of debate whether melanoma cells can switch between high and low metastatic phenotype reversibly while containing melanin pigment. If so, we believe that elastic properties of melanoma cells may play a key role in determining the efficiency of cells to spread in vivo. The mechanical effect of melanin granules on the transmigration abilities of melanoma cells is a novel observation, which has not been reported by other researchers. We view this as an important discovery considering that the current concepts of melanoma metastasis ignore the mechanical effects of melanin granules on melanoma invasiveness (56). We believe that the results obtained in this study will contribute to better understanding of the process of metastasis of malignant melanoma and may help to identify new cellular factors that determine the invasive abilities of melanoma cells. The results may also have a significant impact on future melanoma research. It should be emphasized that the majority of studies are carried out using non-pigmented melanoma cells. This is mostly due to the fact that melanoma cells lose the ability to synthesize melanin under in vitro conditions. However, taking into consideration the magnitude of the effect observed in this study together with the fact that melanin synthesis is an inherent property of melanoma cells in vivo, we can conclude that melanin pigmentation in melanoma cells should no longer be omitted, particularly when investigating melanoma metastasis. The correlation between the number of melanin granules in melanoma cells and their metastatic abilities could have a significant impact on future melanoma diagnosis. As a highly aggressive tumor, melanoma once diagnosed, is immediately excised and sent to histopathology. Although such analysis can determine whether cancer cells have already begun to invade the surrounding tissues, it is not quantitative and cannot provide information on how many cells are capable of undergoing metastasis. We believe that quantitative analysis of the melanin content in melanoma cells, obtained from patients with diagnosed melanoma could facilitate simple and accurate determination of the cell metastatic phenotype. Cells, containing more melanin granules, would likely indicate lower invasive potential. This, together with existing diagnostic methods, could lead to a more complete melanoma diagnosis. Such a diagnosis could better assess the risk of developing metastatic tumors, helping to employ optimized treatment. Of course, many additional experiments are necessary before such approach could be tested in a preclinical set-up.
Acknowledgements The authors would like to thank Dr Mariusz Kepczynski for confocal microscopy images and Dr Anna Pilat for help with zymography assay. The authors would also like to thank Barbara Czuba-Pelech for excellent
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technical assistance. This work was supported in part by Poland Ministry of Science and Higher Education (Statutory Activity of the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University – DS 16 and DS 7). Confocal microscopy analysis was carried out with the equipment purchased, thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Grant No. POIG.02.01.00-12-023/08). Cell migration analysis was carried out with the equipment purchased, thanks to the
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financial support of the structural funds from the European Union (Grants No: POIG.02.01.00-12-064/08 and POIG.01.02-00-109/09).
Author contribution M.S. designed the study. M.S. and A.Z. performed the experiments. M.S., A.Z., P.H., Z.M., K.B. and T.S. analysed the data. M.S. and T.S. wrote the paper.
Conflict of interests The authors have declared no conflicting interests.
23 Zhou Z, Zheng C, Li S et al. Nanomedicine 2013: 9: 864–874. 24 Ryszawy D, Sarna M, Rak M et al. Carcinogenesis 2014: 35: 1920–1930. 25 Sarna M, Zadlo A, Koczurkiewicz P et al. Melanin modifies nanomechanical properties of pigmented melanoma cells. In: The Melanocyte and Its Environment. Bologna: Medimond, 2012: 23–27. 26 Sarna M, Zadlo A, Pilat A et al. Pigment Cell Melanoma Res 2013: 26: 727–730. 27 Slominski A, Ermak G, Wortsman J. In Vitro Cell Dev Biol Anim 1999: 35: 564–565. 28 Sarna T, Swartz H N. The physical properties of melanin. In: Nordlund J J, Boissy R E, Hearing V J, King R A, Oetting W S, Ortonne J P, eds. The Pigmentary System, Physiology and Pathophysiology. Oxford: Blackwell Publishing Ltd, 2006: 311–341. 29 Zadlo A, Burke J M, Sarna T. Photochem Photobiol Sci 2009: 6: 830–837. 30 Sarna T, Burke J M, Korytowski W et al. Exp Eye Res 2003: 76: 89–98. 31 Hutter J L, Bechhoefer J. Rev Sci Instrum 1993: 64: 1868–1873. 32 Dufrene Y F. Curr Opin Microbiol 2003: 6: 317– 323. 33 Hermanowicz P, Sarna M, Burda K et al. Rev Sci Instrum 2014: 85: 063703. 34 Radmacher M. Methods Cell Biol 2007: 83: 347–372. 35 Gavara N, Chadwick R S. Nat Nanotechnol 2012: 7: 733–736. 36 Touhami A, Nysten B, Dufrene Y F. Langmuir 2003: 19: 4539–4543. 37 Marshall J. Transwell invasion assay. In: Wells C M, Persons M, eds. Cell Migration: Developmental Methods and Protocols. New Jersey: Humana Press, 2011: 97–110. 38 Sarna M, Wybieralska E, Miekus K et al. Mol Med Rep 2009: 2: 865–871. 39 Madeja Z, Szymkiewicz I, Zaczek A et al. Biochem Cell Biol 2001: 4: 425–440.
40 Snoek-van Beurden P A, Von den Hoff J W. Biotechniques 2005: 38: 73–83. 41 Sealy R C, Hyde J S, Felix C C et al. Science 1982: 217: 545–547. 42 Hu D N, Wakamatsu K, Ito S et al. Melanoma Res 2009: 19: 75–79. 43 Cross S E, Jin Y S, Rao J et al. Nat Nanotechnol 2009: 4: 72–73. 44 Manimaran M, Tay F, Chaw K C. J Phys: Conf Ser 2006: 34: 1143–1147. 45 Fu Y, Chin L K, Bourouina T et al. Lab Chip 2012: 12: 3774–3778. 46 Krause M, Te Riet J, Wolf K. Phys Biol 2013: 10: 065002. 47 Hofmann U B, Westphal J R, van Muijen G N et al. J Invest Dermatol 2000: 115: 337–344. 48 Hofmann U B, Eggert A A, Blass K et al. Cancer Res 2003: 63: 8221–8225. 49 Friedl P, Alexander S. Cell 2003: 147: 992– 1009. 50 Camp J T, Elloumi F, Roman-Perez E et al. Mol Cancer Res 2011: 9: 3–13. 51 Umezu T, Ohyashiki K, Kuroda M et al. Oncogene 2013: 32: 2747–2755. 52 Gupta G P, Massague J. Cell 2006: 127: 679– 695. 53 Pinner S, Jordan P, Sharrock K et al. Cancer Res 2009: 69: 7969–7977. 54 Carreira S, Goodall J, Denat L et al. Genes Dev 2006: 20: 3426–3439. 55 Meredith P, Sarna T. Pigment Cell Res 2006: 19: 572–594. 56 Zbytek B, Carlson J A, Granese J et al. Expert Rev Dermatol 2008: 3: 569–585.
Supporting Information Additional supporting data may be found in the supplementary information of this article: Figure S1. Height images and corresponding profiles of melanoma cells with different number of melanin granules and of non-pigmented control cell.
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Original Article
www.wileyonlinelibrary.com/journal/EXD
Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells Michal Sarna1,2, Andrzej Zadlo2, Pawel Hermanowicz3, Zbigniew Madeja4, Kvetoslava Burda1 and Tadeusz Sarna2 1 Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Krakow, Poland; 2Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 3 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 4Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Correspondence: Michal Sarna, Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Mickiewicza 30, Krakow 30-059, Poland, Tel.: (+48) 12 6172984, Fax: (+48) 12 6340010, e-mail: [email protected]
Abstract: The relationship between melanin pigmentation and metastatic phenotype of melanoma cells is an intricate issue, which needs to be unambiguously determined to fully understand the process of metastasis of malignant melanoma. Despite significant research efforts undertaken to solve this problem, the outcomes are far from being satisfying. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells. This prompted us to examine the mechanical effects of melanosomes on the transmigration abilities of melanoma cells. Here, we show for the
first time that melanin granules inhibit transmigration abilities of melanoma cells in a number of granules dependent manner. Moreover, we demonstrate that the inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may play a key role in the efficiency of melanoma cells spread in vivo. Our findings may also contribute to better understanding of the process of metastasis of malignant melanoma.
Introduction
knowledge would contribute to better understanding of metastasis of melanoma and could even lead to the development of a more advanced diagnosis of this malignant tumor. Metastasis is a complex process in which cancer cells spread from the primary tumor to colonize distant organs (11). One of the most critical steps in the process of metastasis is the invasion of host tissues (12). During this stage of metastasis cancer cells encounter multiple barriers inside the tissues (13,14), with the most significant being the endothelial barrier (15). To pass through different barriers cancer cells must be able to undergo extensive deformation of their cellular body (16). This unique mechanical property of cancer cells depicted as elasticity is a result of significant changes in the cytoskeleton of the cells and is acquired during cancer transformation (17,18). Consequently, cancer cells become very soft and highly deformable, which is believed to be one of the main reasons for their ability to transmigrate through multiple barriers highly efficiently (19,20). Different studies have demonstrated that cancer cells with lower values of the Young’s modulus (the measure of elasticity) also exhibit an increased invasive potential (21–24). Therefore, cell elasticity is viewed as an important indicator of the metastatic phenotype of cancer cells. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells (25,26). This has led us to formulate the hypothesis that melanosomes could significantly affect the transmigration abilities of melanoma cells and, as a result, they could affect the cells invasiveness. Because of obvious importance, the hypothesis
Among cellular properties of different cancer types, the ability of melanoma to synthesize melanin clearly stands out. Melanin in pigmented cells, including melanoma cells is present in the form of distinct granules called melanosomes (1). These organelles display a range of different shapes and sizes (2) and have unusual mechanical properties (3). Melanoma originates from transformed melanocytes – cells that normally produce melanin in the skin (4). However, unlike melanocytes, which continuously transfer the synthesized pigment to keratinocytes, effectively decreasing the number of melanosomes inside the cells; melanoma cells do not excrete the pigment, which may lead to heavy pigmentation of the cells (5). This unique property of melanoma cells puzzled researchers for decades, and still remains unclear. Under normal conditions melanin synthesis is regulated by different factors and has protective function (6). On the other hand, when melanogenesis is deregulated (such as in melanoma), it can have a detrimental effect (7–9). In spite of significant research efforts, the relationship between melanin pigmentation and metastatic phenotype of melanoma cells remains controversial. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. It is important to realize that melanoma is one of the deadliest types of cancer with poor prognosis when diagnosed in the metastatic stage (10). Therefore, it is of key importance to determine the relationship between melanin pigmentation and metastatic phenotype of melanoma cells. Such
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
Key words: cell elasticity – invasion – melanin pigmentation – melanoma – metastatic phenotype
Accepted for publication 3 August 2014
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should be verified experimentally. It is important to realize that mechanical effects of melanin granules on melanoma cells invasive abilities have not been studied by other researchers and remain unknown. In this work, we show for the first time that melanin granules inhibit the transmigration abilities of melanoma cells through artificial barriers in vitro. Moreover, we demonstrate that the inhibitory effect of melanosomes depends on the number of granules in the cells. On the other hand, our data indicate that the pigment granules have no significant impact on vital functions of pigmented cells including their tumorigenic properties. This has led us to conclude that the observed inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may be an important indicator of the metastatic phenotype of melanoma cells in vivo. Moreover, our findings may contribute to better understanding of the process of metastasis of malignant melanoma.
Methods Cell culture Human melanoma SKMEL-188 cells were cultivated in Ham’s F10 culture medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and maintained at 37°C in a 5% CO2 humidified atmosphere. Under these conditions SKMEL-188 cells did not synthesize melanin and such cells were used in the analysis as control non-pigmented melanoma cells. Different levels of melanin pigmentation of the cells were obtained using a non-invasive method for melanin synthesis as described elsewhere (27). After this protocol, cells were passaged and maintained in Ham’s F10 culture medium for 48 h to ensure that cells did not synthesize melanin during the analysis and to minimize the risk of any shock to the cells caused by the exchange of culture media. All experiments described in this work were carried out in Ham’s F10 medium.
Melanin determination in cell samples Analysis of melanin content in the examined samples was determined by electron paramagnetic resonance (EPR) spectroscopy based on intensity and spectral parameters of characteristic EPR signals of eumelanin and pheomelanin (28). For EPR analysis, cells were detached from the culture dish, pelleted, counted, incubated in high concentration of zinc acetate, frozen and stored at 77 K. The number of cells for each sample was approximately 106 cells and final concentration of zinc ions was 50 mM. EPR examination was carried out in liquid nitrogen, using standard finger-type quartz dewar and EMX-AA spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at X-band with 100 kHz magnetic modulation. Detailed description of EPR analysis used in this work can be found elsewhere (29,30). The average number of melanin granules inside cells was determined by comparing double integrals of the EPR signals of cell samples with that of 2.05 mg/ ml synthetic cystein-L-dopa-melanin used as a standard and with the EPR signal of a known number of purified melanosomes isolated from pigmented SKMEL-188 cells run under identical experimental conditions. Based on relative intensities of these signals, an equivalent EPR signal of a single melanin granule was determined. Such standard was then used to calculate the average number of melanin granules per cell in cell samples. The number of melanin granules used as a standard in EPR analysis was approximately 2 9 108 granules.
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Elasticity analysis Mechanical analysis of the cells was conducted using Agilent 5500 atomic force microscope (AFM) (Agilent Technologies, Austin, TX, USA) coupled with top-down optics (Navitar, Rochester, NY, USA). During the analysis, cells were maintained in culture medium at 37°C. AFM measurements were performed using standard silicon nitride cantilevers (Bruker Probes, Karlsruhe, Germany) with nominal tip radius of 20 nm and with experimentally determined spring constants (31). Mechanical measurements were conducted in force spectroscopy mode that consists of measuring force-displacement curves (32). Force curves were collected at a rate of 1 Hz per curve from a ‘grid’ of 8 9 8 force curves applied onto the centre area of a cell. Before each grid was selected, a topography image of a cell was obtained in tapping ac mode, to precisely position the grid onto the centre of the cell body. During mechanical measurements, both the applied force and indentation depth were carefully monitored to prevent damage to the cells and to avoid possible substrate induced effects. Forty cells for each sample were analysed. Furthermore, to show a co-localization effect of melanin granules on the elastic properties of the cells, representative force maps of pigmented and non-pigmented cells were made. This was achieved from a grid of 128 9 128 force curves applied onto the entire scan area. Analysis of force curves was made using AtomicJ – our Open Source software (33). Briefly, the values of the Young’s modulus were estimated from force curves by converting force-displacement curves into force-indentation curves and fitting with the Sneddon model (34). During the analysis, the correction for thin samples was used to minimize any substrate generated artifacts (35). The half opening angle of the AFM tip was 25°, and the Poisson ratio of the cells was taken to be 0.5, which is typical for soft biological materials (36).
Transwell invasion assay Transmigration efficiency of the cells was determined using a well established Transwell assay for the analysis of the invasive abilities of cancer cells as described elsewhere (37). To highlight the mechanical aspect of the experiment, the membrane was neither coated with collagen and endothelial cells nor with Matrigel. For the analysis, cells were seeded into 6-well plates (three wells per condition) containing Transwell inserts (pore size: 8 lm; Corning Incorporated, Corning, NY, USA). After 24 h, cells were trypsinized and counted. The number of cells transmigrated through porous membrane was compared with the number of cells that remained on the top of the membrane. The assay was repeated three times for statistical analysis.
Proliferation analysis For proliferation analysis, cells were seeded into 12-well plates (one plate per condition) and maintained in culture for 24, 48, 72 and 96 h. After each time interval, cells were trypsinized and counted. The number of cells determined after 24 h for each experimental condition was the reference number of cells indicated as 100%. The assay was repeated three times for statistical analysis.
Migration analysis Migration activity of the cells was determined based on time-lapse monitoring of the movement of individual cells as described elsewhere (38). This was conducted using a fully automated Leica DMI6000 B (Leica Microsystems GmbH, Wetzlar, Germany)
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Melanin granules inhibit melanoma invasion
inverted microscope with a dedicated incubation chamber. During the analysis, cells were maintained in culture medium at 37°C in a 5% CO2 humidified atmosphere. The movement of the cells was recorded for 8 h, with 5 min time intervals. Tracks of individual cells were determined from a series of changes in the cell centroid positions as described elsewhere (39). Trajectories of 40 cells for each condition were analysed for statistics.
Gelatin zymography assay Expression of matrix metalloproteinase was determined using standard protocol for gelatin zymography as described elsewhere (40). Prior to the analysis, cells were incubated in serum-free culture medium for 24 h, after which the supernatants were collected and equivalent amounts of protein for each sample were diluted in sample buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol and 0.4% bromophenol blue). Samples were electrophoresed on SDS-PAGE gels containing 1% gelatin. After electrophoresis, gels were washed in 2.5% Triton X-100 for 30 min to remove SDS. Subsequently, gels were placed in substrate buffer (50 mM Tris pH 7.7, 5 mM CaCl2 and 0.02% NaN3) and incubated for 40 h at 37°C under gentle agitation. After incubation, gels were stained in 40% methanol, 7% glacial acetic acid solution containing 0.4% Coomassie Brilliant Blue R250 for 45 min and destined in the same solution without Coomassie Brilliant Blue for 30 min. The assay was repeated on cell samples from three independent cultures. Densitometric analysis of the gel bands was made using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Results Figure 1 shows EPR spectra of human melanoma cells containing different amounts of melanin pigment and non-pigmented melanoma cells. These results clearly demonstrate that only pigmented cells (Fig. 1a–c) exhibit strong EPR signal with magnetic parameters typical for melanin free radicals (41). The pigment in these cells is a mixture of pheomelanin and eumelanin, which is common for human melanoma cells (42). On the other hand, no EPR signal of melanin radicals is detected in non-pigmented melanoma cells (Fig. 1d). Figure 1 also shows EPR spectra of synthetic cysteine-L-dopa-melanin and purified melanin granules isolated from melanoma cells used as standards in EPR analysis (Fig. 1e and f). The average number of melanin granules inside cells determined by EPR spectroscopy is shown in Table 1. Figure 2 shows results obtained with AFM and demonstrate the impact of melanin granules on the elastic properties of melanoma cells. These results show histograms of Young’s modulus values for cells containing different number of melanin granules (Fig. 2a–c) and non-pigmented melanoma cells (Fig. 2d). As evident from the data, cells containing more melanosomes exhibit higher values of the Young’s modulus. Moreover, the log-normal distribution of Young’s modulus values for pigmented cells becomes more pronounced as the number of melanosomes inside cells increases. Similar effect was observed in our previous studies
Confocal microscopy Immunofluorescence analysis was performed on cells fixed with 3.7% formaldehyde, permeabilised with 0.1% Triton X-100, blocked with 3% BSA and stained with mouse monoclonal antibody against human microtubule, followed by Alexa Fluor 488 goat anti-mouse IgG, with TRITC-phalloidin and Hoechst 33342 (Sigma-Aldrich), respectively. Images were obtained using Nikon Ti-E A1 inverted microscope coupled with laser scanning module equipped with: 405, 488, 561 and 638 nm laser diodes, 4 PMT detectors for fluorescence and 1 PMT detector for transmission (Nikon Instruments, Tokyo, Japan). Images were analysed using NIS-Elements AR 3.1 software (Nikon).
Statistical analysis Statistical significance of differences in mean values was assessed using two-sample independent Student’s t-test at the 95% confidence level. Differences among means were reported using approximated P values. Statistical analysis was made using Mathematica 8.0 software.
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Figure 1. Determination of melanin content inside cells. Electron paramagnetic resonance (EPR) spectra of melanoma cells with different amounts of melanin pigment (a–c) and non-pigmented control cells (d). The EPR signal of melanin in pigmented cells is a superposition of a pheomelanin signal (lower field component) and an eumelanin signal (higher field component). EPR spectra of synthetic cysteine-L-dopa-melanin (e) and purified melanin granules isolated from melanoma cells (f) that were used as standards in the determination of the average number of melanin granules per cell in cell samples. Signal intensities (y-axis) were normalized to maximum amplitude to better show the contribution of pheomelanin component in the spectra.
Table 1. Transmigration efficiency coupled with elastic properties of human melanoma cells versus number of melanin granules inside the cells Degree of cell pigmentation
Number of melanin granules1
Young’s modulus (kPa)2
Non-pigmented Lightly pigmented Moderately pigmented Heavily pigmented
– 22 2 42 3 73 4
1.97 3.23 4.91 6.97
0.05 0.85* 1.22** 1.56***
Transmigration efficiency (%)3 18.79 13.83 9.18 3.94
0.51 0.76* 0.46** 0.27***
Statistical significance between mean values for both Young’s modulus and transmigration efficiency is depicted as the following: *statistically significant versus control cells, **statistically significant versus lightly pigmented cells and ***statistically significant versus moderately pigmented cells. For all values P < 0.0001. Average number of melanin granules per cell (mean SD) determined by EPR spectroscopy. 2 Average values of Young’s modulus (mean SD) determined based on function fit to the data in the histograms. 3 Transmigration efficiency represents the average number of cells (mean SD) transmigrated through porous membrane determined based on a Transwell assay (n = 9 for each sample). 1
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Figure 2. Impact of melanin granules on the elastic properties of melanoma cells. Histograms of Young’s modulus values for cells with different number of melanin granules: (a) 22 2 melanosomes, (b) 42 3 melanosomes, (c) 73 4 melanosomes and non-pigmented control cells (d) no granules were determined in this sample. Data for pigmented cells were fitted with log-normal functions whereas data for non-pigmented cells was fitted with Gaussian function. Forty cells for each condition were analysed with 64 measurement points on a single cell (n = 2560 for every sample). Representative atomic force microscope (AFM) amplitude images of pigmented (e) and non-pigmented (f) melanoma cells and corresponding force maps of Young’s modulus values (g, h). Insets in the images show optical preview of the cells during AFM analysis. Scale bars in the images represent 10 lm. Colour bars in force maps represent Young’s modulus values, range: 0–50 kPa.
(25,26) and discussed in detail. Numerical values of Young’s modulus together with the number of melanin granules inside cells are shown in Table 1. It should be emphasized that during AFM analysis, elasticity measurements were taken from the central region of the cell to avoid possible artifacts when going ‘away’ from the centre of the cell towards cell edges (43). The cell central body is also the most critical area of cancer cells when considering transmigration trough artificial barriers in vitro or different barriers in vivo. This is because the cell central body (the thickest part of a cell) must undergo significantly higher deformation, when compared to outer regions of the cell such as thin lamellipodia (44–46). Figure 2 also shows representative AFM images (Fig. 2e and f) and corresponding force maps (Fig. 2g and h) of pigmented and non-pigmented melanoma cells, respectively. Insets in the images clearly show that melanin granules surround the nucleus of a pigmented cell. As evident from force maps, the values of the Young’s modulus are significantly higher around the cell nucleus (at the locations of the melanosomes) when compared to non-pigmented control cell. It should be emphasized that melanin granules did not modify the topography of pigmented cells when compared to non-pigmented cells. Figure S1 shows height images and corresponding profiles of melanoma cells with different number of melanosomes and non-pigmented melanoma cells. As evident from the data, pigmented and non-pigmented cells exhibit similar topography. Transmigration efficiency of the cells determined from a Transwell assay is shown in Table 1. These results demonstrate that cells containing more melanin granules are less likely to transmigrate through membrane pores compared to cells without melanosomes. The data clearly shows that melanin granules exhibit an inhibitory effect on the transmigration abilities of human melanoma cells and that the effect depends on the number of granules inside the cells. To determine whether the observed inhibitory effect of melanosomes on the transmigration abilities of melanoma cells was solely mechanical in nature, we examined the impact of melanin
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Figure 3. Impact of melanin granules on melanoma cell vital functions and the cell cytoskeleton. (a) Column graph showing proliferation abilities of pigmented and non-pigmented melanoma cells. Results obtained from three independent experiments (n = 9 for each sample). (b) Column graph showing migration activity of pigmented and non-pigmented melanoma cells based on the movement of individual cells (n = 40 for each sample). (c) Representative gel is from three zymographies carried out on independent cell cultures (n = 3 for every sample) and shows the expression of pro-MMP-9 for pigmented and non-pigmented melanoma cells. Numbers on the gel refer to the level of melanin pigmentation of the cells: 0 – non-pigmented control cells, 1 – lightly pigmented, 2 – moderately pigmented and 3 – heavily pigmented. Representative confocal microscopy images of actin (red), microtubules (green) and nucleolus (blue) for pigmented (d) and nonpigmented (e) melanoma cells. Overlay images of scattered laser light and fluorescence for pigmented (f) and non-pigmented (g) melanoma cells show melanin granules in pigmented cell (indicated with arrows) surrounding the cell nucleus. No granules are present in non-pigmented cell. Scale bars in the images represent 10 lm.
granules on the cell vital functions. Figure 3 shows the impact of melanosomes on proliferation abilities (Fig. 3a), followed by migration activity (Fig. 3b) and expression of metalloproteinase (Fig. 3c) in pigmented and non-pigmented melanoma cells. Based on quantitative analysis of the cell proliferation abilities and migration activity, we can conclude that melanin granules inhibit neither proliferation nor motility of pigmented melanoma cells in any significant way when compared to non-pigmented melanoma cells. Moreover, densitometric analysis of gels obtained from zymography assay revealed no significant differences in the expression levels of pro-matrix metalloproteinase-9 (pro-MMP-9) between cells containing different number of melanin granules and non-pigmented control cells (data not shown). Although human melanoma cells can express different matrix metalloproteinases (47), the MMP-9 is viewed as one of the most important metalloproteinases engaged in the formation of metastatic tumors (48). Figure 3 also shows the impact of melanin granules on the cell cytoskeleton. Representative fluorescence images of actin and microtubules for pigmented and non-pigmented melanoma cells are shown in Fig. 3d and e. These images reveal that melanin granules do not modify the cell cytoskeleton in any significant way. Overlay images of scattered laser light and fluorescence for pigmented and non-pigmented melanoma cells, respectively (Fig. 3f and g), clearly show that melanin granules in pigmented cell are mostly localized around the cell nucleus. We believe that such distribution of melanin granules inside melanoma cells is one the main reasons of the reduced capability of pigmented melanoma cells to pass through narrow barriers. The hard, nondeformable melanosomes (3) that are present in the most critical
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area of melanoma cells prevent the cells to undergo extensive deformation of their cellular body necessary for passage through small membrane pores. As evident from elasticity measurements taken from the cell central region, cells containing melanin granules show higher values of the Young’s modulus, which translates to reduced deformation capabilities of the cells when compared to non-pigmented control cells. Thus, in pigmented melanoma cells, melanin granules act as a natural inhibitor of the cells transmigration abilities.
Discussion In this work, we have demonstrated for the first time that melanin granules inhibit the abilities of melanoma cells to transmigrate through narrow barriers in vitro, in a number of granules dependent manner. Moreover, we have determined that the inhibitory effect of melanosomes is mechanical in nature being due to a dramatic change in the elastic properties of the cells caused by the presence and distribution of the pigment granules inside the cells. It is important to stress that the presence of melanosomes in melanoma cells did not affect any of the cell vital functions, including their proliferation activity, their motile abilities and expression of the key metalloproteinase (MMP-9). All these factors play a crucial role in the process of metastasis (49). Therefore, the results obtained in this study support the validity of our experimental model for melanoma cells invasive abilities, that is the clean, uncovered version of a Transwell assay. It should be stressed that the main purpose of our work was to demonstrate the mechanical aspect of the observed phenomenon in a quantitative manner. This goal would be very difficult to achieve, if possible at all, using a membrane coated with both collagen and endothelial cells or coated with Matrigel. The latter models are more suitable to examine other properties of cancer cells important for invasion as demonstrated elsewhere (50,51). Our experiments employing melanoma cells with different numbers of melanin granules allowed us to analyse, in non-invasive manner, cells with different elastic properties and, to correlate the elasticity of the cells with their transmigration efficiency. The obtained results confirm the widely accepted paradigm that elasticity is a unique indicator of the invasive abilities of different cancer cells, including melanoma cells. There is no doubt that elasticity is just one of many properties of cancer cells that play an important role during metastasis (52). However, taking into consideration the magnitude of the effect observed in this study together with the fact that melanoma cells can contain large numbers of melanosomes (5); we can conclude that at least under some circumstances, elasticity of melanoma cells could be the dominant factor responsible for the efficient spreading of the cells inside host tissues. Recently, using intra-vital imaging Pinner et al. demonstrated that pigmented melanoma cells displayed in vivo a lower metastatic phenotype then non-pigmented melanoma cells. Moreover, the authors reported that the cells could switch between high and low metastatic phenotype. Such switch was accompanied by changes in the cells pigment state (53). The authors attributed the observed results to the level of cell differentiation. Hence, pigmented melanoma cells are considered to be more differentiated and therefore less invasive (54). However, in their experimental model the authors used different growth factors and stimuli for the induction or inhibition of melanin pigmentation in cells. It cannot be ruled out that the used factors had a signif-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
icant impact on the level of cells differentiation. It should be emphasized that in our study, we did not use any growth factors or stimuli that would enhance the level of cells differentiation in any significant way. This allowed us to obtain melanoma cells with different amount of melanin pigment and to correlate the level of cell pigmentation with their transmigration abilities. It is also important to realize that melanin is not susceptible to enzymatic degradation (55), and the only way to reduce its content in melanoma cells is to stop synthesis of melanin. With continuous divisions, such cells will become less pigmented. However, this depigmentation process takes time and is not instantaneous. It is a matter of debate whether melanoma cells can switch between high and low metastatic phenotype reversibly while containing melanin pigment. If so, we believe that elastic properties of melanoma cells may play a key role in determining the efficiency of cells to spread in vivo. The mechanical effect of melanin granules on the transmigration abilities of melanoma cells is a novel observation, which has not been reported by other researchers. We view this as an important discovery considering that the current concepts of melanoma metastasis ignore the mechanical effects of melanin granules on melanoma invasiveness (56). We believe that the results obtained in this study will contribute to better understanding of the process of metastasis of malignant melanoma and may help to identify new cellular factors that determine the invasive abilities of melanoma cells. The results may also have a significant impact on future melanoma research. It should be emphasized that the majority of studies are carried out using non-pigmented melanoma cells. This is mostly due to the fact that melanoma cells lose the ability to synthesize melanin under in vitro conditions. However, taking into consideration the magnitude of the effect observed in this study together with the fact that melanin synthesis is an inherent property of melanoma cells in vivo, we can conclude that melanin pigmentation in melanoma cells should no longer be omitted, particularly when investigating melanoma metastasis. The correlation between the number of melanin granules in melanoma cells and their metastatic abilities could have a significant impact on future melanoma diagnosis. As a highly aggressive tumor, melanoma once diagnosed, is immediately excised and sent to histopathology. Although such analysis can determine whether cancer cells have already begun to invade the surrounding tissues, it is not quantitative and cannot provide information on how many cells are capable of undergoing metastasis. We believe that quantitative analysis of the melanin content in melanoma cells, obtained from patients with diagnosed melanoma could facilitate simple and accurate determination of the cell metastatic phenotype. Cells, containing more melanin granules, would likely indicate lower invasive potential. This, together with existing diagnostic methods, could lead to a more complete melanoma diagnosis. Such a diagnosis could better assess the risk of developing metastatic tumors, helping to employ optimized treatment. Of course, many additional experiments are necessary before such approach could be tested in a preclinical set-up.
Acknowledgements The authors would like to thank Dr Mariusz Kepczynski for confocal microscopy images and Dr Anna Pilat for help with zymography assay. The authors would also like to thank Barbara Czuba-Pelech for excellent
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technical assistance. This work was supported in part by Poland Ministry of Science and Higher Education (Statutory Activity of the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University – DS 16 and DS 7). Confocal microscopy analysis was carried out with the equipment purchased, thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Grant No. POIG.02.01.00-12-023/08). Cell migration analysis was carried out with the equipment purchased, thanks to the
References
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financial support of the structural funds from the European Union (Grants No: POIG.02.01.00-12-064/08 and POIG.01.02-00-109/09).
Author contribution M.S. designed the study. M.S. and A.Z. performed the experiments. M.S., A.Z., P.H., Z.M., K.B. and T.S. analysed the data. M.S. and T.S. wrote the paper.
Conflict of interests The authors have declared no conflicting interests.
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Supporting Information Additional supporting data may be found in the supplementary information of this article: Figure S1. Height images and corresponding profiles of melanoma cells with different number of melanin granules and of non-pigmented control cell.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 813–818
